Cancer is the first or second leading cause of death before the age of 70 years in the majority of countries worldwide [1]. The incidence and mortality of cancer is affected by multiple factors, including lifestyle, the type of cancer and its specificity, stage of cancer, mode of treatment, etc. ^[2][3][2,3]. Thus, the efficacy of treatment varies and the need for personalized therapy [4], the development of a novel therapy development ^[5][6][7][8][5,6,7,8], and the search for novel anticancer drugs [9] play a crucial role. One of the most serious problems that significantly reduces the therapeutic effectivity in cancer treatment is the phenomenon of multidrug resistance (MDR). The theory of MDR is not novel; initially it was prevalently associated with the reduction of chemotherapy efficacy [10] but it is now widely known that the phenomenon of MDR can also significantly reduce the therapeutic effectivity of other treatment approaches, even photodynamic therapy (PDT) [11]. The mechanism of MDR is largely associated with the ATP-binding cassette (ABC) transporters [12] with broad substrate specificity, which includes many therapeutics and photosensitizers (PSs), too ^{[13][14][15][16][17][18][19][20][21][22][23]}[13,14,15,16,17,18,19,20,21,22,23]. Therefore, restricting substrate specificity and bypassing the efflux of target agents represent one of the possible solutions for limiting MDR. The concept of a dynamic nanoplatform using non-biodegradable nanoparticles (NPs) to permanently retain PSs has been established on just this base and it has been progressively developed in the last fifteen years ^{[24][25][26][27][28][29][30][31][32][33][34][35][36]}[24,25,26,27,28,29,30,31,32,33,34,35,36]. When we talk about nanotechnology, we consider a scale—an order of magnitude—of size, or length. The prefix ‘nano-’ is derived from the Greek word nannos, meaning “very short man”. In scientific units ‘nano’ is used to denote one-billionth of the base unit. Nanotechnology includes the formation and use of materials, structures, devices, and systems that have unique properties because of their small size [37]. The term ‘nanotechnology’ can be dated back to 1974 when it was first used by Norio Taniguschi. Taniguschi described nanotechnology as the technology that forms materials at the nanometer level [38]. Nanomaterials and NPs, the nanometer-sized objects, are the leading edge of the rapidly developing field of nanotechnology and have great applicability in biology and medicine. As NPs are much smaller in size than the cells of living organisms, they are suitable for bio tagging and labeling, drug or gene delivery, diagnosis and detection of specific proteins or pathogens, etc. In general, simple NPs are made from a single material, whereas composite and core/shell NPs are composed of two or more materials. The core itself can consist of several functional layers, allowing the use of nanomaterials in multifunctional approaches. The core particle is usually surrounded and protected by another outer layer or by several layers (a shell) that are composed of some inert material, organic molecules, or biocompatible materials. However, specific linker molecules, ligands, and additional layers are more often conjugated on the surface of NPs in order to improve and add some useful properties, and to increase the biocompatibility of the nanomaterial ^[39][40][39,40]. Technological progress makes it possible to create novel materials, modify the characteristics of currently created materials, or prepare some multimaterial structures. Novel technologies enable multifunctional nanoplatforms to be constructed with enhanced targeting to the particular sites of the tumor mass. In 2015, Yang et al. [41] were the first to introduce multifunctional chemo-PDT and fluorescent imaging systems based on mesoporous silica NPs. Subsequently, many types of NPs and PSs were analyzed for the purpose of improving the therapeutic efficacy of PDT and chemotherapy, not only against the multidrug resistant cancer cells ^{[41][42][43][44][45][46][47][48][49][50][51][52][53][54][55][56][57]}[41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57], but also against the cancer stem cell phenotype ^{[58][59][60][61]}[58,59,60,61].

2. Nanoparticles—General Systematization

Over the last twenty years, great progress has been made in the field of NP development and their utilization can be found in a huge number of therapeutic approaches ^[62][63][62,63]. Generally, NPs are defined as submicroscopic particles with a size range from 1 to 100 nm [64]. Many refined review papers discussing the systematic classification, description of preparation methods, and their complex physical and biochemical characterization of NPs have been published ^[64][65][64,65]. However, there are several important applications of nanomaterials, and there is no doubt that material engineering represents one of the most progressive scientific areas. The development of novel materials is also substantial ^{[47][48][66][67][68]}[47,48,66,67,68], and the validity and completeness of any systematic nomenclature related to the systematization of NPs is therefore temporary. Generally, NPs are naturally occurring or chemically prepared synthetic materials. Initially, NPs are very often categorized as active or passive; being active means that they carry active surface moieties ^[69][70][71][69,70,71]. Lucky et al. (2015) presented a classification system based on the functions or tasks of NPs, namely in PDT. According to the system, NPs are divided into three classes: carriers of PSs, PSs by themselves, and energy transducers of PSs. Currently, the first class of NPs, having the role of PS carriers, is composed of biodegradable and non-biodegradable NPs. The group of biodegradable NPs is represented by polyester and polyacrylamide NPs, liposomal NPs, dendrimer-based NPs, and natural macromolecule-based NPs that are presented by albumin. The class of nonbiodegradable NPs is composed of silica, gold, and magnetic NPs.

3. Problematic Attributes and Limitations of PDT

The era of potential cancer treatment using modern PDT started more than sixty years ago ^[72][130]. Since then, research in this field has been developing rapidly and is considered to be a very efficient modality for the treatment of various malignant and non-malignant diseases. The selective destruction of cancer cells with minimal toxicity towards non-cancer cells represents a significant advantage for their successful application in clinical use ^[73][131]; however, the improvement of the targeting characteristics of PDT is still crucial ^[74][132]. The therapeutic efficacy and success of PDT is based on three fundamental components—the properties of the PS, the spectral characteristics of light and its output power, and finally, the presence of molecular oxygen ^[75][133]. In comparison with conventional therapeutic approaches like chemotherapy or radiotherapy, PDT is not an invasive method, which consequently reduces the risk of infections and brings excellent cosmetic results ^[76][134]. PDT has also shown its applicability in the treatment of microbial ^[77][135] and viral infections ^[78][79][80][136,137,138]. Moreover, it is possible to use it to treat actinic keratosis, superficial, nodular basal cell carcinoma, Bowen´s disease, and some types of viral skin infections ^[76][134]. At the molecular level, the effect of PDT depends mainly on singlet oxygen ^[75][133]. The molecules of the PSs must be placed close to the targeted organelles at the time of irradiation as the half-life time of singlet oxygen (<0.04 µs) and the radius of its action (<0.02 nm) are short ^[81][139]. The sites that are most preferred for the accumulation of PSs are mitochondria, lysosomes, plasma and intracellular membranes, Golgi apparatus, and the endoplasmic reticulum. Controversially, accumulation in the cell nucleus is very rare ^[82][140]. Besides, the cell nucleus is not a preferred target of PDT because it can potentiate mutagenesis under certain conditions as a consequence of genetic material effects [55]. In general, intracellular damage of mitochondria and the endoplasmic reticulum is prevalently associated with apoptosis, whereas PDT targeted on lysosomes or the plasma membrane increases the possibility of necrosis ^[83][141]. Thus, the PSs that accumulate close to the mitochondria or endoplasmic reticulum have a higher application potential. It is clear that the allocation of PSs within the tissue and cells has a great impact on the outcome of PDT. Firstly, the distribution of PSs among organelles depends on the transport efficiency of the PS molecules into the intracellular environment. However, the previously mentioned aggregate formation of PS molecules significantly limits its uptake and reduces the efficiency of PDT ^[84][85][82,142]. Thus, the search for novel PS solvents represents one of the essential lines of investigation in PDT research ^[86][83]. Furthermore, the systemic administration of drugs leads to their unwanted interaction with the surrounding environment. Therefore, poor penetration is not a terminally limiting factor that restricts the clinical use of many PSs. These interactions could also decrease or even fully reduce a desired pharmaceutical effect ^[87][143], which has been observed in the case of neutral leuko-methylene blue molecules, where the cationic reduction of methylene blue molecules resulted from their systematic application ^[88][89][90][144,145,146]. The higher accumulation rate of PSs observed outside the neoplastic section of the tumor mass or even in healthy tissues and skin is associated with their damage after irradiation and could contribute to tumor development ^[74][132]. The distribution kinetics of PS molecules ^[91][92][147,148] or, more precisely, the molecular mechanisms affecting their influx and efflux cell characteristics are probably the fundamental factors modulating the status of PS accumulation in particular tissues or cells ^[93][149]. Besides the accumulation of PSs, another important factor affecting the treatment efficacy or failure is the phenomenon of MDR. The concept of MDR is not novel, as it has been very extensively studied over the last few decades ^{[10][94][95][96]}[10,150,151,152]. Initially, only the reduction of chemotherapy efficacy was attributed to MDR [10], but since the 1990s, there has been a growing body of evidence highlighting the fact that MDR exceeds the borders of chemotherapy and could affect other therapeutic approaches, even PDT. Currently, the mechanism of MDR is greatly associated with the overexpression of ABC transporters, and MDR-associated protein-1 (MRP1/ABCC1), breast-cancer-resistant protein (BCRP/ABCG2), and P-glycoprotein (P-gp/ABCB1) have been the most extensively studied representatives [5]. In physiological conditions, ABC membrane transporters fulfill an irreplaceable role in the transport of toxic molecules out of the intracellular space using the energy from ATP hydrolysis. This mechanism prevents the intracellular accumulation of toxic compounds and protects the cells from damage ^[97][153]. A higher expression of these efflux pumps has been observed for example in the intestine, blood–brain barrier, and blood–testis barrier ^[98][154]. ABC transporters have also been observed in other internal organs, such as the liver and kidney, where they take part in detoxification ^[99][155]. Their presence in the placenta ^[100][156] is associated with the protection of the fetus from toxic factors in the maternal circulation ^[101][157]. Interestingly, the significant expression of ABCG2 transporter has been observed in the cell membranes of hematopoietic progenitor cells and other stem cells where their presence is linked with the proliferation and maintenance of the stem cell phenotype. In cancer cells, the expression of ABCG2 is related to the presence of “side population” (SP) phenotype. The SP cells are resistant to certain chemotherapeutic drugs, thanks to their higher efflux activity. Moreover, the SP fraction actively supports tumor formation and its progression ^[102][158]. Due to the fact that ABCG2 is standardly expressed in stem cells, it has been suggested that it may also serve as one of the possible, but not universal ^[103][159], biomarkers of CSCs ^[104][160]. ABC transporters show a broad substrate specificity, including many therapeutic drugs and PSs, too. In 1994, Kessel et al. [12] identified copper benzochlorin iminium salt (CDS1) as a substrate of P-gp, and other PSs molecules have since been confirmed as substrates of P-gp, such as tetrabromorhodamine 123 [13], thiorhodamins, and selenorhodamins [14]. Additionally, protoporphyrin IX [16], hematoporphyrin IX [17], pheophorbide a [17], 2-[1-hexyloxyethyl]-2-devinyl pyropheophorbide-a ^[16][18][16,18], phytoporphyrin (phylloerythrin) [19], chlorin e6 [17], benzoporphyrin derivative monoacid ring A [17], hypericin ^[20][105][20,161], and iminoacridine ^[21][22][21,22] have been identified as substrates of ABCG2. Besides the fact that many PSs are substrates of ABC transporters, they can also actively modulate the level of certain efflux pumps. Indeed, some recently published papers have detected an increased expression of BCRP in the lung cancer cell line A549 ^[103][159] or elevated BCRP and MRP1 levels after hypericin application in dark conditions in colorectal HT-29 ^[15][20][15,20], and ovarian A2780 and A2780cis cell lines [23]. Moreover, Jendželovská et al. [23] observed an enhanced MRP1 expression in A2780 and A2780cis cells only 6 h after treatment with 0.5 µM hypericin. In HT-29 cells, the elevated expression of MRP1 was observed even 16 h after the application of 0.1 µM hypericin concentration ^[15][20][15,20]. Jendželovský et al. (2019) stated that the elimination of hypericin from cancer cells represents one of the essential obstacles affecting the efficacy of PDT with hypericin (HY-PDT). The decreased intracellular level of PSs affected by BCRP were associated with a lower therapeutic efficacy of PDT, which was also observed in other PSs, such as protoporhyrin ^{[17][18][106][107][108]}[17,18,162,163,164], chlorin e6 ^[17][109][17,165], pheophorbide ^[110][166], pyropheophorbide a ^[111][167], pyropheophorbide a methyl ester [17], pheophorbide a ^[112][168], 2-(1-hexyloxethyl)-2-devinil pyropheophorbide-a (HPPH, Photochlor) [18], benzoporphyrin derivative monoacid ring A (BPD-MA, Verteporfin) [18], aminolevulonic acid-protoporhyrin IX (ALA-PpIX) ^[113][169], and photofrin (PT) ^[114][170].

4. Nanoparticles as a Possible Solution for Reducing the MDR Effect in Cancer Treatment

As mentioned in the section above, the phenomenon of MDR represents a very serious, if not the most important, factor that significantly reduces the efficacy of PDT. However, the problem is even more complex because, as mentioned above, many PSs are not only the substrates of ABC transporters, but could even enhance the MDR effect via upregulating their expression. All things considered, the lower therapeutic effect of PDT is the consequence of cascade reactions, where the enhanced amount of ABC transporters limits the intracellular accumulation of PSs. The lower therapeutic efficacy accompanied by survival of the targeted cell fraction can result in tumor regrowth and higher malignancy, which was observed using in vivo experimental models ^{[74][91][115][116][117]}[132,147,171,172,173]. Finally, the tumorigenic potential, which was characterized by the enhanced ability to repopulate the tumor, is a typical feature of CSCs ^[118][174]. Thus, novel medical approaches focusing on the reduction of the MDR mechanism could make significant progress in cancer treatment. With this in mind, biodegradable natural or synthetic NPs carrying PSs were initially utilized for PDT, with polyester- and polyacrylamide-based NPs; liposomal NPs belong to the most extensively studied representatives of this category. The analyses with tetanus toxoid prepared in liposomes clearly showed a greater antibody response in comparison with free toxoid. Moreover, after the repeated application of free toxoid, the experimental animals died. In contrast, the animals who were treated with toxoid prepared in liposomes preserved good health ^[119][78]. Later, multiple liposome modifications were analyzed to improve the membrane stability ^[120][175] and entrapment potential for a wide range of molecules like chemotherapeutics ^[121][176], PSs ^{[122][123][124][125][126][127]}[177,178,179,180,181,182], or mRNA ^[128][129][183,184]. Interestingly, thanks to long-term research, alongside the COVID-19 pandemic situation, liposomes have been used as transporters in officially approved mRNA vaccines ^[130][185]. The data have shown that the utilization of biodegradable NPs could significantly improve solubility, the effectivity of PSs delivery ^{[122][123][124]}[177,178,179], tumoricidal activity ^[123][178], wavelength absorption parameters of PSs, the PS accumulation ratio between the skin and the tumor, and the tumor regression potential ^[125][180], as well as their long-storing capability ^[126][181]. Moreover, Lima et al. (2013) showed that the utilization of lipid NPs with a core, stabilized by the surfactant known as solid lipid NPs (SLNs), significantly reduces the essential deficiencies of the conventional lipid NPs linked with the low entrapment efficiency of the PSs. Importantly, the structural modification did not induce the toxic or phototoxic effect in vitro. In relation to SLNs, the entrapment efficiency of hypericin was more than 80% higher ^[127][131][182,186]. In addition, using HEp-2 human larynx carcinoma cells, B16-F10 mouse melanoma cells ^[127][182], and Hep G2 human hepatocellular carcinoma cells ^[131][186], a higher absorption effectivity, higher photostability, lower photodegradation ^[127][131][182,186], more effective singlet oxygen production, and about 30% higher hypericin intracellular accumulation and 26% higher phototoxicity (in comparison to the experimental group treated with free hypericin) were detected. Thus, SLNs might help to partially overcome the enhanced efflux of PSs by transporter proteins, which is the typical manifestation of MDR, by increasing the intracellular PS content ^[127][182]. On the contrary, there are several pieces of evidence pointing to the fact that the higher PS encapsulation efficiency observed in SLNs ^{[127][131][132]}[182,186,187] or polyactic acid polymeric NPs (PLA) has a negative effect on their photoactivity. Surprisingly, Zeisser-Labouebe (2006) observed a lowered photocytotoxic effect of encapsulated hypericin when compared to free hypericin on NuTu-19 cells, depending on the increasing encapsulation efficacy of PLA. The influence of drug loading on the phototoxic effect of biodegradable NPs could be explained by multiple parameters. The most likely explanation lies in the NP size, where particles with a diameter higher than 200 nm could significantly lower PDT effectivity as a consequence of their decreased permeability, and thus limit access to the tumor ^[131][133][186,188]. Another potential reason could be that PSs loaded into NPs with a smaller diameter may be closer to the surface of the NPs, and a more rapid release is therefore possible ^[132][187]. Observations where a higher drug loading capacity is paradoxically associated with the limited drug release capability of NPs are not only noted in relation to PSs. Mu and Feng (2003) observed a similar trend with the utilization of paclitaxel-loaded poly(DL-lactide-co-glycolide) (PLGA) NPs with a diameter about 400 nm, and Görner et al. (1999) clearly showed that larger NPs exhibit a slower release ^[134][135][189,190]. Using lidocaine loaded in poly(d,l-lactic acid) NPs varying in particle size from about 250 to 820 nm, they also suggested that the release profile of NPs is affected by a combination of the size and drug loading parameters of the NPs. The authors also suggested the creation of a heterogenous matrix with a higher drug loading in the NPs whose presence limits drug release. Therefore, the loaded drug must firstly be dissolved in these highly loaded NPs, which causes its slower release. In relation to PSs, the use of larger NPs (>200 nm) ^[131][186] could be associated with a higher rate of aggregate formation in these NPs, which could significantly restrict the photocytotoxic effect of PDT ^[132][187]. Naturally, biodegradable NPs are designed to load, deliver, and release particular molecules. Therefore the major drawbacks of biodegradable delivery systems are associated with the risk of PS efflux by the MDR mechanisms ^[87][143], and also with the persistence of the post-treatment accumulation of drugs in the skin and eyes, resulting in long-term phototoxic side effects ^[136][191].